The present invention relates generally to methods for manipulating the production of flavonoids in plants by manipulating endogeneous and incorporated gene activity in the flavonoid biosynthetic pathway and compositions for use in such methods. In particular, it relates to methods for increasing flavonoid levels by altering the level of chalcone isomerase activity. Chalcone isomerase is an enzyme involved in the biosynthetic pathway of flavonoids.
Flavonoids form a large group of polyphenolic compounds, based on a common diphenylpropane skeleton, which occur naturally in plants. Included within this class of compounds are flavonols, flavones, flavanones, catechins, anthocyanins, isoflavonoids, dihydroflavonols and stilbenes. The flavonoids are mostly present as glycosides.
In tomato fruits, the main flavonoid found is naringenin chalcone (Hunt et al, Phytochemistry, 19, (1980), 1415-1419). It is known to accumulate almost exclusively in the peel and is simultaneously formed with colouring of the fruit. In addition to naringenin chalcone, glycosides of quercetin and, to a lesser extent, kaempferol are also found in tomato peel.
Reports in the literature suggest that there is increasing evidence that flavonoids are potentially health-protecting components in the human diet. Epidemiological studies suggest a direct relationship between cardioprotection and increased consumption of flavonoids, in particular flavonols of the quercetin and kaempferol type, from dietary sources such as onion, apples and tea (see, for example, Hertog et al, Lancet, 342 (1993), 1007-1011).
Flavonoids have been reported to exhibit a wide range of biological activities in vitro including anti-inflammatory, anti-allergic and vasodilatory activity (Cook et al, Nutritional Biochemistry, 7, (1996), 66-76). Such activity has been attributed in part to their ability to act as antioxidants, capable of scavenging free radicals and preventing free radical production. Within this group of compounds, those having the most potent antioxidant activity are the flavonols (Rice-Evans et al, Free Radical Research, 22, (1995), 375-383). In addition, flavonoids can also inhibit the activity of key processes such as lipid peroxidation, platelet aggregation and capillary permeability (see Rice-Evans et al, Trends in Plant Science, 2, (1997), 152-159).
Based on studies of this type, there is presently considerable interest in the development of food products from plants rich in such protective flavonoids.
It would be desirable to produce plants which intrinsically possess elevated levels of health protecting compounds such as flavonoids in order to develop food products with enhanced protective properties. Traditionally, the approach to improving plant varieties has been based on conventional cross-breeding techniques, but these are slow as they require time for breeding and growing successive plant generations. More recently, recombinant DNA technology has been applied to the general problem of modifying plant genomes to produce plants with desired phenotypic traits. Whilst reference has been made in the literature to the use of genetic manipulation techniques in modifying the flavonoid biosynthetic pathway, as discussed beneath, it is notable that these attempts have been directed in general towards modifying pigmentary anthocyanin production.
The flavonoid biosynthetic pathway is well established and has been widely studied in a number of different plant species (see, for example, Koes et al, BioEssays, 16, (1994), 123-132). Briefly, three molecules of malonyl-CoA are condensed with one molecule of Coumaroyl-CoA, catalysed by the enzyme chalcone synthase, to give naringenin chalcone which rapidly isomerises, catalysed by chalcone isomerase, to naringenin. Subsequent hydroxylation of naringenin catalysed by flavanone 3-hydroxylase leads to dihydrokaempferol. Dihydrokaempferol itself can be hydroxylated to produce either dihydroquercetin or dihydromyricetin. All three dihydroflavonols subsequently can be converted to anthocyanins (by the action of dihydroflavonol reductase and flavonoid glucosyltransferase) or alternatively converted to flavonols such as kaempferol, quercetin and myricetin by the action of flavonol synthase.
A schematic overview of the flavonoid biosynthetic pathway is presented in appendix 1, FIG. 1.1.
The manipulation of flavonoid levels in plants by altering the expression of a single flavonoid biosynthetic gene is disclosed by Napoli (1990, Plant Cell, 2:279-289). Napoli discloses the introduction of a chimeric chalcone synthase (CHS) gene into Petunia. Said introduction is described to result in a block in the anthocyanin biosynthesis. The resulting transformed petunia plants therefore contained lower levels of flavonoids than untransformed plants, presumably due to co-suppression of the endogeneous CHS activity.
Que (1997, Plant Cell, 9: 1357-1368) discloses a comparison of the effect of strong and weak promoters that drive sense chalcone synthase transgenes in large populations of independently transformed plants. It is shown that a strong transgene promoter is required for high frequency cosuppression of CHS genes and for the production of a full range of phenotypes.
Howles (1996, Plant Physiol. 112: 1617-1624) discloses the stable genetic transfer of the flavonoid biosynthetic gene phenylalanine ammonia-lyase (PAL) from french bean into tobacco. A proportion of the obtained transgenic tobacco plants is shown to display overexpression of PAL activity. According to Howles PAL overexpressing plants do not contain altered levels of flavonoids.
It has been disclosed by Tanaka et al (1995, Plant and Cell Physiology 36: 6, 1023-1031) that heterologous transformation of dihydroflavonol reductase (DFR) can be used for the production of plants with altered levels of anthocyanins.
There is no disclosure in the literature of the manipulation of flavonoids in plants by means of overexpression of chalcone isomerase.
Accordingly, there remains a continuing need for the development of methods for enhancing the levels of flavonoids, in particular flavonols, in plants.
Therefore, in a first aspect, the invention provides a method for producing a plant capable of exhibiting altered levels of flavonoids comprising incorporating into said plant one or more gene sequences encoding a protein with chalcone isomerase activity, or incorporating a nucleotide sequence encoding a protein functionally equivalent thereto.
The invention also provides a plant having one or more transgenes each encoding a protein with chalcone isomerase activity, or a protein functionally equivalent thereto, incorporated into its genome such that its ability to produce flavonoids is altered.
According to a highly preferred embodiment, the invention further provides a tomato plant having one or more transgenes each encoding a protein with chalcone isomerase activity, or a protein functionally equivalent thereto, incorporated into its genome such that its ability to produce flavonoids is altered.
Also provided is a transformed plant having enhanced flavonoid levels, not being chalcones, particularly enhanced flavonol levels compared to similar untransformed plants. Preferably the level of said flavonoids, not being chalcones, in transformed plants is at least 4 times higher than in similar untransformed plants, more preferred 5-100, most preferred 10-40 times higher than in similar untransformed plants.
Further provided is a fruit-bearing plant, particularly a tomato plant, having flavonoids, particularly flavonols, in the peel of the fruit.
Seeds, fruits and progeny of such plants and hybrids are also included within the invention.
The invention further provides DNA constructs coding for a protein with chalcone isomerase activity, or a functionally equivalent sequence of said DNA construct, operably linked to a promoter.
When transformed into a plant cell, these constructs are useful for overexpressing genes encoding proteins with chalcone isomerase activity, thereby altering the ability of the plant to produce flavonoids. The invention also provides for plants comprising these constructs together with seeds, fruits and progeny thereof.
Food products such as sauces, dressings, ketchups and soups, comprising at least part of a plant prepared according to the invention are also provided.
Also provided are skin and hair protective products comprising at least part of a plant according to the invention.
Also provided are pharmaceuticals comprising at least part of a plant according to the invention.
As used herein, xe2x80x9cplantxe2x80x9d means a whole plant or part thereof, or a plant cell or group of plant cells. It will be appreciated that also extracts are comprised in the invention.
A xe2x80x9cflavonoidxe2x80x9d or a xe2x80x9cflavonolxe2x80x9d may suitably be an aglycon or a conjugate thereof, such as a glycoside, or a methyl, acyl, sulfate derivative.
A xe2x80x9cprotein with chalcone isomerase activityxe2x80x9d is a protein being capable of enzymatically catalysing the conversion of a chalcone into a flavanone, for example narichalcone into naringenin.
A xe2x80x9cgenexe2x80x9d is a DNA sequence encoding a protein, including modified or synthetic DNA sequences or naturally occurring sequences encoding a protein, and excluding the 5xe2x80x2 sequence which drives the initiation of transcription.
A xe2x80x9cDNA sequence functionally equivalent theretoxe2x80x9d is any sequence which encodes a protein which has similar functional properties.
According to another embodiment, a functionally equivalent DNA sequence shows at least 50% similarity to the respective DNA sequence. More preferably a functionally equivalent DNA sequence shows at least 60%, more preferred at least 75%, even more preferred at least 80%, even more preferred at least 90%, most preferred 95-100% similarity, to the respective DNA sequence.
According to the most preferred embodiment a functionally equivalent DNA sequence shows not more than 5 base pairs difference to the respective DNA sequence, more preferred less than 3, e.g. only 1 or 2 base pairs different.
According to another embodiment a functionally equivalent sequence is preferably capable of hybridising under low stringent conditions to the respective sequence.
xe2x80x9cBreakerxe2x80x9d is the ripening stage corresponding to the appearance of the first flush of colour on the green fruit.
xe2x80x9cOperably linked to one or more promotersxe2x80x9d means the gene, or DNA sequence, is positioned or connected to the promoter in such a way to ensure its functioning. The promoter is any sequence sufficient to allow the DNA to be transcribed. After the gene and promoter sequences are joined, upon activation of the promoter, the gene will be expressed.
A xe2x80x9cconstructxe2x80x9d is a polynucleotide comprising nucleic acid sequences not normally associated in nature.
An xe2x80x9calteredxe2x80x9d level of flavonoids is used throughout this specification to express that the level of specific flavonoids in the transformed plants differs from the level of flavonoids present in untransformed plants. Preferably the difference is between 0.1 and 100 fold. It will be appreciated that the specific flavonoids as meant here are flavonoids other than chalcones as said specific flavonoids are formed at the expense of chalcones.
Therefore in the specification where these flavonoids are meant reference will be made to xe2x80x9cspecific flavonoidsxe2x80x9d.
An xe2x80x9cincreasedxe2x80x9d level of flavonoids is used to indicate that the level of is preferably at least 4 times higher than in similar untransformed plants, more preferred 5-100, most preferred 10-40 times higher than in similar untransformed plants.